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Applications of microfluidics in chemical biologyDouglas B Weibel1 and George M Whitesides2
This review discusses the application of microfluidics in
chemical biology. It aims to introduce the reader to
microfluidics, describe characteristics of microfluidic systems
that are useful in studying chemical biology, and summarize
recent progress at the interface of these two fields. The review
concludes with an assessment of future directions and
opportunities of microfluidics in chemical biology.
Addresses1 Department of Biochemistry, University of Wisconsin-Madison,
433 Babcock Drive, Madison, WI 53706, USA2 Department of Chemistry and Chemical Biology, Harvard University,
12 Oxford Street, Cambridge, MA 02138, USA
Corresponding authors: Weibel, Douglas B ([email protected])
and Whitesides, George M ([email protected])
Current Opinion in Chemical Biology 2006, 10:584–591
This review comes from a themed issue on
Model systems
Edited by Amit Basu and Joel Schneider
Available online 23rd October 2006
1367-5931/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2006.10.016
Introduction‘Microfluidics’ refers to the science and technology of
manipulating fluids in networks of channels with dimen-
sions of �5–500 mm; its history has been the subject of a
recent review by Whitesides [1��]. Microfluidic systems
transport volumes of fluid that vary from microliters
(10�6 l) to femtoliters (10�15 l) in channels that are
usually embossed in the surface of a polymer, but are
occasionally fabricated in glass (Figure 1). Soft lithogra-
phy is a set of techniques used frequently to produce
microfluidic systems, and is based on embossing channels
in a thin slab of a polymer; poly(dimethylsiloxane)
(PDMS) is the material most commonly used in academic
laboratories [2,3]. The techniques of soft lithography
provide an inexpensive and convenient alternative to
conventional methods of microfabrication based on laser-
or e-beam writing and photolithography in glass or silicon.
The use of soft lithography also makes possible the
fabrication of prototype microfluidic systems in short
periods of time (typically less than one day), and the
generation of multiple copies of a device in several hours.
PDMS has several attractive properties for the fabrication
of microfluidic channels for use with aqueous systems of
Current Opinion in Chemical Biology 2006, 10:584–591
biochemicals and cells: it is soft, flexible, biocompatible,
electrically insulating, unreactive, transparent to visible
and ultraviolet light, permeable to gases, and only mod-
erately permeable to water. Its surface can be oxidized
easily to present primarily Si-(OH) groups; this reaction
makes PDMS hydrophilic, and, more importantly, allows
PDMS to be sealed to other polymers, and to glass,
typically without an adhesive. Many of these character-
istics make PDMS particularly useful for studying biolo-
gical systems, such as cells and small organisms [4]. The
primary disadvantages of PDMS as a material for micro-
fluidics are that it absorbs a range of organic solvents and
compounds (particularly alkyl and aryl amines), and that
its surface properties can be difficult to control [5]. Roll-
and et al. have demonstrated recently that cross-linked
perfluoropolyether elastomers have many physical prop-
erties in common with PDMS but do not absorb many of
the organic solvents that PDMS does; sealing layers of
this polymer to itself or other materials is more difficult
than sealing PDMS using the oxidative route [6].
Microfluidics has several features that have attracted users
in biology, chemistry, engineering and medicine. It
requires only small volumes of samples and reagents,
produces little waste, offers short reaction and analysis
times, is relatively cheap, and has reduced dimensions
compared with other analytical devices. In addition, micro-
fluidics offers structures with length scales that are com-
parable to the intrinsic dimensions of prokaryotic and
eukaryotic cells, collections of cells, organelles, and the
length scale of diffusion of oxygen and carbon dioxide in
tissues (every cell, in vivo, is no more than�100 mm from a
capillary). These characteristics make microfluidics parti-
cularly useful in studying biology and biomedicine [7].
The number of applications of microfluidics in biology,
analytical biochemistry, and chemistry has grown as a
range of new components and techniques have been
developed and implemented for introducing, mixing,
pumping, and storing fluids in microfluidic channels.
Despite rapid progress in this area, there remain several
unsolved problems: preparing and introducing samples;
interfacing microfluidic channels with the human hand;
working with a range of sample volumes (e.g. Vacutainers,
a drop of blood, a biopsy sample, a single cell); and
portability.
Characteristics of microfluidics withrelevance to chemical biologyMicrofluidics is an enabling technology. It makes possible
the exploration of biological systems — from molecules,
through cells, to small, multicellular organisms such as
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Applications of microfluidics Weibel and Whitesides 585
Figure 1
Images of (a,b) microfluidic channels, (f,g) microfluidic components and (c,d,e,h) microfluidic devices. (a) Laminar streams of solutions of dye (in
water) flowing in a microfluidic channel. The fluid is flowing from the six channels on the left into the central channel on the right where flow is laminar.
Reproduced from [43] with permission. Copyright 2005, ACS. (b) An image of a section of the Topaz Dynamic Array for high-throughput protein
crystallization. The integrated fluidic circuit contains microfluidic chambers, channels, and pumps. Image courtesy of Fluidigm Inc. (http://
www.fluidigm.com). (c) A microfluidic chemostat in which an intricate system of plumbing is used to grow and study bacteria. Reproduced from [9�]
with permission. Copyright 2005, AAAS. (d) A microdiluter system in which two fluids are repeatedly split at a series of nodes, combined with
neighboring streams, and mixed. At the end of the network of channels, the streams of fluid carrying different concentrations of green and red dye
are combined and produce a gradient. Reproduced from [19] with permission. Copyright 2001, AAS. (e) A linear gradient of fluorescein produced
using the microdiluter shown in (d). Reproduced from [19] with permission. Copyright 2001, AAS. (f) An image of metal wires fabricated 10 mm away
from a microfluidic channel (40 mm diameter). The metal structures have a variety of uses — for example, as electromagnets for producing
electromagnetic fields in adjacent microchannels. Image courtesy of A Siegel (unpublished data). (g) Microfluidic channels for directing the
movement of cells of swimming bacteria. Image courtesy of W DiLuzio (unpublished data). (h) A combinatorial tool based on two layers of PDMS
microfluidic channels bonded together at right angles to each other, and separated by a thin membrane. Reproduced from [22] with permission.
Copyright 2001, ACS.
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586 Model systems
Caenorhabditis elegans — in ways that are not possible using
larger systems. Below we provided a summary of the
characteristics of microfluidics that are particularly useful
in probing biological systems using small molecules.
Miniaturization
Microfluidic systems allow small numbers of cells, or even
single cells, to be isolated, manipulated, and examined
[8�,9�,10]. The microchannels in microfluidic systems
provide a mechanism for introducing nutrients and
reagents, exchanging media, and removing waste from
cells or small samples of tissue. Single cells can be isolated
physically in microfluidic systems while keeping them in
fluidic contact; this controlled contact allows them to
exchange small molecules, growth factors and other pro-
teins, and thus allows studies of signaling and toxicology
on small scales [11�,12�] (Figure 2).
Small volume, large surface area
As the physical dimensions of channels decrease, their
surface-to-volume ratio increases, and the rates of heat
and mass transfer increase due to steeper gradients in
Figure 2
A microfluidic system for engineering cell–cell contact. (a) An image of a mi
membrane contact. The dashed line highlights the area of the device illustra
were trapped. The blue and orange spheres represent cells. (c) The top cell
cell contained no dye. In the absence of membrane–membrane contact betw
(d) When the two cells were brought into membrane–membrane contact, th
Current Opinion in Chemical Biology 2006, 10:584–591
temperature and concentration. Large surface areas can,
in some cases, be beneficial (e.g. in gas exchange), or
detrimental (e.g. in the non-specific adsorption of pro-
teins from solution onto the walls of the container).
Several techniques minimize the non-specific adsorption
of proteins and other biomolecules on the walls of micro-
fluidic channels. For example, a wide range of studies
have identified oligomers of ethylene oxide (poly(ethy-
lene glycol); PEG) as coatings that reduce the adsorption
of proteins and cells on surfaces [13,14]. Huang et al.describe another approach for preventing the non-specific
adsorption of proteins to PDMS channels by coating the
walls with n-dodecyl-b-D-maltoside [15].
An important characteristic of working with small fluidic
systems is that the volumes of samples, reagents and cells
that are needed are quantitatively small. Minimizing
waste can be particularly important when working with
biologically hazardous material. Lee et al. described a
particularly relevant example: the multistep synthesis of
an 18F-labeled probe in a microfluidic system for PET
scanning [16�].
crofluidic system for bringing pairs of mammalian cells in membrane–
ted in (b). (b) A cartoon of the region of the device in which cells
was loaded with an intracellular fluorescent dye (calcein AM); the bottom
een the cells, dye was not transferred from the top cell to the bottom cell.
e transfer of dye occurred. Reproduced from [12�] with permission.
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Applications of microfluidics Weibel and Whitesides 587
Scaling out
Microfluidics facilitates a characteristic that we refer to as
‘scaling out’: that is, thousands of identical microfluidic
structures can be produced in an area that is several
square centimeters; these structures make it possible to
carry out many experiments in parallel. A relevant exam-
ple are microfluidic chips produced by Thorsen et al. that
have an area of 5 cm2 and contain a network of 256
microchambers (each with a volume of �750 pl) that
are addressed individually using integrated pneumatic
valves [17]. We believe that scaling out is of increasing
importance in biology as the field moves toward quanti-
tative data because it allows many parallel experiments to
be performed on cells or organisms under identical con-
ditions, and thus provides data that will allow a mean-
ingful estimate of the reproducibility and reliability of
biological measurements.
Automation
The integration of techniques for introducing samples,
pumping, storing, mixing and metering out fluids in
microfluidic systems is beginning to lead to the miniatur-
ization of laboratory instruments. Automation is used in
many integrated microfluidic devices, particularly in sys-
tems in which pneumatic valves pump fluids, colloids and
cells into addressable chambers where reactions or ana-
lyses are carried out [17]. Automated techniques for
distributing reagents in parallel microfluidic channels
compete with expensive liquid handling robots that dis-
pense fluids in 96-, 384- or 1536-well plates.
Laminar flow
The Reynolds number (Re) is a dimensionless quantity
that describes the behavior of fluids, and is defined by
Re = rvd/m, where r is the density of fluid (g/cm3), v is the
velocity of the fluid (cm/s), d is the hydraulic diameter
(cm) of the channel, and m is the viscosity of the fluid (g/
cm�s). Below a Reynolds number of �2000, flow is lami-
nar; above this value the flow is turbulent. In channels
with a surface-to-volume ratio characteristic of microflui-
dic systems, the flow of fluid is dominated by viscous
dynamics and is laminar. Adjacent streams of miscible
fluids flowing through microchannels flow side-by-side,
with mixing only by diffusion at the interface of the
streams. Several groups have exploited this characteristic
to produce stable gradients of small molecules, proteins
and growth factors [18�,19,20,21��]. These gradients are
typically perpendicular to the flow of fluid, can be pro-
duced on surfaces or in solution, and have temporal and
spatial stability.
Applications of microfluidics in chemicalbiologyMicrofluidic systems are beginning to appear in a suffi-
ciently wide range of applications that it is not practical to
survey them completely in a limited space; a recent set of
articles in a Nature Insight section on microfluidics provides
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a good, recent overview over this area [1��]. Here, we have
selected several recent papers that we think will be of
interest to the community of chemical biologists, and
organized them into categories; we summarize them below.
Arrays
Microfluidic systems consisting of crossed sets of micro-
channels provide a way of studying the interaction of large
numbers of molecules with proteins or cells in a combi-
natorial layout. Ismagilov et al. described a combinatorial
tool based on two layers of PDMS microfluidic channels
bonded together at right angles to each other, and sepa-
rated by a thin membrane (Figure 1h) [22]. The membrane
permitted chemical contact between the two layers of
channels and kept small particles from crossing the two
streams. This system was used in assays for pathogenic
microorganisms, but, more generally, it offers the oppor-
tunity to conduct a wide range of different types of assays
(for example, antibody-based assays for anti-gp120 for
AIDS) requiring the formation of precipitates. In principle,
this kind of device should make it possible to examine
several samples of serum for the presence of proteins.
Gradients
The manipulation of laminar streams of fluids (Figure 1a)
in microfluidic channels makes it possible to create gra-
dients of almost arbitrary complexity of small molecules,
growth factors and other proteins in solution and on
surfaces. Methods that use a common attachment scheme
based on biotin and avidin are particularly well developed
[18�]. Microfluidics enables the formation of gradients
that cannot be generated using other techniques, includ-
ing very steep gradients of concentration that extend over
several orders of magnitude, gradients with complicated
profiles, and gradients of gradients [18�,19,20,21��,23,24]
(Figure 1e).
Several groups have studied the behavior and differentia-
tion of cells in response to chemical signals in channels.
One of the earliest examples of interactions between cells
and chemical gradients was by Jeon et al., who studied the
chemotaxis of human neutrophils on a gradient of inter-
leukin-8 in a microfluidic channel [25]. Other groups have
studied the migration and behavior of neutrophils on
gradients of proteins [20,26]. Pihl and co-workers used
a microfluidic system to profile molecules for pharmaco-
logical activity [24]. The authors created an integrated
device in which gradients of drugs were produced and
their activity against voltage-gated K+ ion channels was
measured (at different concentrations) by patch clamping
individual CHO cells exposed to different regions of the
gradient. Chung et al. created gradients of growth factors
to study the growth and differentiation of human neural
stem cells [27]. Gunawan and co-workers studied the
migration and polarity of rat intestinal cells on gradients
of extracellular matrix proteins in microfluidic channels
[28].
Current Opinion in Chemical Biology 2006, 10:584–591
588 Model systems
Figure 3
A microfluidic system for positioning small molecules with subcellular
resolution. (a) A cartoon depicting the treatment of selected regions of
mammalian cells with reagents using laminar streams of fluid. (b) An
image of a bovine capillary endothelial cell treated with Mitotracker Red
CM-H2XRos (left side of cell) and Mitotracker Green FM stain (right side
of cell); the nucleus was stained with the DNA-binding dye, Hoechst
33342. Reproduced with permission from [37].
Laminar flow can also be used to create gradients in
temperature. Luccetta et al. created a thermal gradient
in a microfluidic channel whose dimensions were
designed to study the effect of temperature on the
development of an embryo of Drosophila melanogasterby exposing different parts of the same embryo to fluids
at two different temperatures [29�].
Microdiluters
Microfluidic diluters (‘microdilutors’) are systems in
which solutions or liquid reagents are carried through a
series of controlled dilutions, and then used in assays
(Figure 1d) [19,30]. These dilutors perform some of the
functions of 96-well plate assays, but use smaller quan-
tities of reagents, and are less labor-intensive. Microdi-
luters that perform multiple cycles of dilution are the
microfluidic version of a multiwell plate. Microdiluters
are just beginning to be used in biochemistry: for exam-
ple, Jiang has used a microdiluter to carry out a miniatur-
ized, parallel, serially diluted immunoassay for analyzing
multiple antigens [31].
Gel structures
One useful property of soft lithography is that it can be
used to form microstructures in gels, or can be used in
conjunction with gels. Microfluidic systems fabricated in
agar, agarose and calcium alginate form biocompatible
structures that can be infused with small molecules,
nutrients or proteins. Cabodi et al. fabricated microfluidic
systems in alginate, and studied mass transfer in the
channels [32�]. Khademhosseini et al. captured fibroblasts
in ‘corrals’ molded in poly(ethylene glycol) within micro-
fluidic channels for high-throughput screening [33]. Gel
structures can serve as containers in which cells can be
grown in the presence of small molecules that diffuse
through the gel structure. Takeuchi and co-workers used
slabs of agarose with embossed microfluidic chambers for
growing cells of Escherichia coli in the presence of small
molecules that alter the phenotype of cells [11�]. Camp-
bell et al. have studied reaction-diffusion systems invol-
ving cleverly designed gels fabricated using soft
lithography; although these methods have not been used
in biochemical systems, their potential in these systems is
obvious [34].
Droplets
Several different microfluidic systems have been
described for producing droplets of liquid (typically
10–500 microns in radius) dispersed in a second liquid.
Droplets produced in microchannels can, in appropriately
designed droplet-generators, be almost monodisperse,
and have a volume that may be as small as several
picoliters; such droplets have been used in a range of
applications. For example, they serve as liquid bioreactors
for encapsulated cells [35�]. Plugs of fluids with nanoliter
volumes form the basis of systems for screening condi-
tions for biochemical reactions and the crystallization of
Current Opinion in Chemical Biology 2006, 10:584–591
proteins. Chen and Ismagilov have reviewed this area
recently [36].
Painting cells
Microchannels provide a convenient mechanism for treat-
ing cells — or portions of cells — with soluble reagents
carried in laminar flows with interfaces smaller than the
dimensions of a cell. Takayama et al. exposed selected
regions of mammalian cells to fluids containing fluores-
cent dyes (that is, ‘painted’ them) by positioning cells at
the interface of two different streams of fluids flowing at
low Reynolds number [37] (Figure 3). The authors used
this technique to: i) study the movement of populations of
mitochondria in cells; ii) disrupt actin filaments in cells;
iii) detach selected regions of cells from surfaces; and iv)
create gradients of small molecules in single cells [38].
Studies of single cells
As we mentioned briefly above, single cells can be iso-
lated and manipulated in microfluidic systems. This
characteristic has been used to study the biochemistry
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Applications of microfluidics Weibel and Whitesides 589
and biophysics of single cells. Wu et al. constructed
microfluidic compartments with picoliter volumes for
carrying out chemical cytometry on single Jurkat T cells
[39]. Cai and co-workers measured the expression of b-
galactosidase in single cells of E. coli in a microfluidic
system with single molecule sensitivity [40��].
Devices
Much of the work in polymeric microfluidic systems has
focused on the design and fabrication of components, and
their combination into devices. There is now a substantial
level of competence in the choice of materials and in the
design of pumps, valves, filters, and the myriad of other
components that must be available to fabricate complex
systems. New work has used co-fabrication (the fabrica-
tion of all of the microsystems needed for a device at one
time) to make an on-chip light source, tunable dye laser,
magnetic separators, heaters, and other sophisticated
components. Related methods have been used to fabri-
cate varieties of cell- and sperm-sorters, and micro-var-
iants of continuous stirred-tank reactors for microbiology
[8�,9�,41]. The rapid advance in the physical science and
technology of microfluidics is the product of a growing
community of researchers, of which some of the names
not so far mentioned or cited are Klavs Jensen, Seth
Freden, Chris Chen, David Weitz, Piotr Garstecki,
Howard Stone, John Wikswo, Paul Yager and others. It
is now time to begin to apply these systems and devices to
real problems in biochemistry; this work is just beginning.
Conclusion and future directionsMicrofluidics brings the advantages of small volumes of
fluids, reagents, and waste, and small numbers of cells, to
chemical studies on biological systems. The techniques
of soft lithography are within the reach of biologists and
biochemists, and make it possible to prototype micro-
fluidic structures rapidly. Public foundries — facilities
designed to fabricate microfluidic devices, typically using
soft lithography — have been established at several
universities (Harvard, California Institute of Technology,
and University of Washington) to eliminate most of the
technically difficult steps in fabricating microfluidic sys-
tems and to extend the technology to the chemical and
biological communities.
Microfluidics is an enabling technology that will make
possible new applications in biological sciences; we
believe it will play an important role in the development
of cell-based assays, single-cell assays, phenotypic
screens, gene-expression profiling, patient-specific assays,
high-throughput and combinatorial methods, and techni-
ques and devices appropriate in cost and performance for
bioanalytical detection in developing economies, in home
health care, and by first responders.
The future of microfluidics offers a range of interesting
and new opportunities. The most important is the appli-
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cation of existing technology to problems in biochemistry
and biomedicine. Beyond that, however, there are several
new opportunities in learning: i) to interface microfluidic
systems with microelectronic devices; ii) to carry out new
kinds of experiments involving very large numbers of
experiments run in parallel to measure the reproducibility
of biochemical (or more importantly, cell biological)
experiments [42�]; iii) to carry out specialized types of
biochemical synthesis (e.g. synthesis of 18F-labeled com-
pounds for PET [16�]; and iv) to incorporate optical and
electromagnetic technologies in chip-based devices in an
integrated way for manipulating samples and for their
analysis.
Microfluidics has much to offer the field of chemical
biology. Chemical biology, in return, has an opportunity
to stimulate research in microfluidics by asking questions
that require the development of new capabilities and
materials. The techniques and tools that arise from this
successful merger will broaden our understanding of
biological interactions and may have applications in other
areas of science.
AcknowledgementsWho thank Adam Siegel, Willow DiLuzio, Steve Quake, and FluidigmInc. for providing several of the images used in Figure 1. This work wassupported by grants from the National Institutes of Health (GM065364)and the Defense Advanced Research Projects Agency (DARPA).
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� of special interest
�� of outstanding interest
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Current Opinion in Chemical Biology 2006, 10:584–591
590 Model systems
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9.�
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11.�
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29.�
Lucchetta EM, Lee JH, Fu LA, Patel NH, Ismagilov RF:Dynamics of Drosophila embryonic patterning networkperturbed in space and time using microfluidics. Nature 2005,434:1134-1138.
This paper describes an approach for creating thermal gradients inmicrofluidic systems. The authors used this system to study the tem-perature effect on the Bicoid morphogen gradient in single embryos ofDrosophila melanogaster.
30. Neils C, Tyree Z, Finlayson B, Folch A: Combinatorial mixing ofmicrofluidic streams. Lab Chip 2004, 4:342-350.
31. Jiang X, Ng JMK, Stroock AD, Dertinger SKW, Whitesides GM:A miniaturized, parallel, serially diluted immunoassay foranalyzing multiple antigens. J Am Chem Soc 2003,125:5294-5295.
32.�
Cabodi M, Choi NW, Gleghorn JP, Lee CSD, Bonassar LJ,Stroock AD: A microfluidic biomaterial. J Am Chem Soc 2005,127:13788-13789.
In this paper, the authors describe a microfluidic biomaterial made byembossing channels in calcium alginate, and characterized mass trans-port in the channels.
33. Khademhosseini A, Yeh J, Jon S, Eng G, Suh KY, Burdick JA,Langer R: Molded polyethylene glycol microstructures forcapturing cells within microfluidic channels. Lab Chip 2004,4:425-430.
34. Campbell CJ, Smoukov SK, Bishop KJM, Grzybowski BA:Reactive Surface Micropatterning by Wet Stamping.Langmuir 2005, 21:2637-2640.
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He M, Edgar S, Jeffries GDM, Lorenz RM, Shelby JP, Chiu DT:Selective encapsulation of single cells and subcellularorganelles into picoliter- and femtoliter-volume droplets.Anal Chem 2005, 77:1539-1544.
This paper describes a technique for encapsulating single cells in dropletsof liquid. Cells were positioned at the orifice of a microfluidic dropletgenerator using optical tweezers; as the droplets were pinched off theyencapsulated single cells.
36. Chen DL, Ismagilov RF: Microfluidic cartridges preloaded withnanoliter plugs of reagents: an alternative to 96-well plates forscreening. Curr Opin Chem Biol 2006, 10:226-231.
37. Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE,Whitesides GM: Subcellular positioning of small molecules.Nature 2001, 411:1016.
38. Takayama S, Ostuni E, LeDuc P, Naruse K, Ingber DE,Whitesides GM: Selective chemical treatment of cellularmicrodomains using multiple laminar streams. Chem Biol 2003,10:123-130.
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39. Cai L, Friedman N, Xie XS: Stochastic protein expression inindividual cells at the single molecule level. Nature 2006,440:358-362.
40.��
Wu H, Wheeler A, Zare RN: Chemical cytometry on a picoliter-scale integrated microfluidic chip. Proc Natl Acad Sci USA2004, 101:12809-12813.
This paper describes an integrated microfluidic device for analyzing thechemical contents of single cells. Single Jurkat T cells were isolated,lysed, and analyzed using this technique.
41. Chung Y, Zhu X, Gu W, Smith GD: Takayama. Microscaleintegrated sperm sorter. Methods Mol Biol 2006, 321:227-244.
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Jiang X, Bruzewicz DA, Wong AP, Piel M, Whitesides GM:Directing cell migration with asymmetric micropatterns.Proc Natl Acad Sci USA 2005, 102:975-978.
This paper describes an approach for directing the motility of mammaliancells by directing their polarization on a surface and then releasing themfrom confinement. The authors used a technique for patterning manyindividual polarized cells in parallel and analyzed their movement afterrelease.
43. Weibel DB, Kruithof M, Potenta S, Sia SK, Lee A, Whitesides GM:Torque-actuated valves for microfluidics. Anal Chem 2005,77:4726-4733.
Current Opinion in Chemical Biology 2006, 10:584–591